Rapid Assembly of Pyrrole-Ligated 1,3,4-Oxadiazoles and Excellent Antibacterial Activity of Iodophenol Substituents

Pyrrole-ligated 1,3,4-oxadiazole is a very important pharmacophore which exhibits broad therapeutic effects such as anti-tuberculosis, anti-epileptic, anti-HIV, anti-cancer, anti-inflammatory, antioxidant, and antibacterial activities. A one-pot Maillard reaction between D-Ribose and an L-amino methyl ester in DMSO with oxalic acid at 2.5 atm and 80 °C expeditiously produced pyrrole-2-carbaldehyde platform chemicals in reasonable yields, which were utilized for the synthesis of pyrrole-ligated 1,3,4-oxadiazoles. Benzohydrazide reacted with the formyl group of the pyrrole platforms to provide the corresponding imine intermediates, which underwent I2-mediated oxidative cyclization to the pyrrole-ligated 1,3,4-oxadiazole skeleton. The structure and activity relationship (SAR) of the target compounds with varying alkyl or aryl substituents of the amino acids and electron-withdrawing or electron-donating substituents on the phenyl ring of benzohydrazide were evaluated for antibacterial activity against Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii as representative Gram(–) and Gram(+) bacteria. Branched alkyl groups from the amino acid showed better antibacterial activities. Absolutely superior activities were observed for 5f-1 with an iodophenol substituent against A. baumannii (MIC < 2 μg/mL), a bacterial pathogen that displays a high resistance to commonly used antibiotics.


Introduction
Oxadiazole is a five-membered heterocyclic aromatic compound composed of four structural isomers depending on the positions of two nitrogen atoms relative to an oxygen atom [1]. Among them, 1,3,4-oxadiazole has received intensive attention in the field of medicinal chemistry due to its broad metabolic profile [2][3][4] and in the field of material science for its excellent optoelectronic properties [5][6][7][8]. As an isostere of an amide and an ester, 1,3,4-oxadiazole serves as a promising pharmacophore for the discovery of new drugs exhibiting antimicrobial, anticonvulsant, anti-inflammatory, analgesic, antitumor, antiviral, antihypertensive, and enzyme inhibitory activities [9]. There have been extensive literature reviews on the specific synthetic methods and diverse biological activities of 1,3,4-oxadiazole derivatives [10][11][12].
Motivated by Raltegravir [13,14], an antiretroviral drug used to treat HIV/AIDS, and Zibotentan [15,16], an anti-cancer drug candidate, a number of poly heterocyclic Pyrrole is a very important structural motif in drug discovery projects because of the wide presence of natural, biologically active pyrrole alkaloid products [25,26]; thus, pyrrole-ligated 1,3,4-oxadiazole would be a perfect base structure for the development of potential lead compounds [27]. Considering the efficacy of the procedures of constructing a 1,3,4-oxadiazole ring, 2-pyrrolyl-5-phenyl-1,3,4-oxadizole 1 would be an ideal core structure, achieved either through dehydration from aroylhydrazide [C] or by cyclization from aroylhydrazone [D]. In the benzene ring, the substituent effects of the core structure 1 on antibacterial activity have been reported for the cases of 4,5-dibromopyrrole [28,29] and 4nitropyrrole [30], respectively.
Pyrrole-2-carbaldehydes 2, derived from the conversion of D-ribose with L-amino acids [31], were demonstrated to be useful, sustainable platform chemicals for the construction of highly functionalized poly heterocyclic compounds [32]. Since natural amino acids themselves demonstrate specific biological activities [33][34][35], it was envisioned that 1,3,4-oxadiazoles 1 from pyrrole-2-carbaldehydes 2 with the N-amino acid moiety would be very interesting core structures for the investigation of their biological activities. The effect of the amino acid moiety of 1 on antimicrobial activities was screened first, and the substituent effects on the benzene ring were then investigated for 5 and 6 with some selected amino acid moieties. We found a marginal size effect of the alkyl groups from amino acids (Val and Ile, etc.) and superior antibacterial activity of the iodophenol substituents of pyrrole-ligated 1,3,4-oxadiazoles 5 and 6 against S. aureus and A. baumannii. All the syntheses of pyrrole-ligated 1,3,4-oxadiazoles 1, 5, and 6 and their antibacterial activities are closely described herein.

Results and Discussion
Structure and activity relationships (SARs) for pyrrole-ligated 1,3,4-oxadiazoles 1 were generally studied by changing substituent groups in the aromatic rings [28][29][30]. We were interested in the SAR of 1 by N-alkyl substituents because pyrrole-2-carbaldehydes 2, the starting materials for 1,3,4-oxadiazoles 1, are easily prepared from L-amino acids, and each amino acid has its own biological activity. Ten L-amino acids with hydrogen, alkyl, aralkyl, ester, and sulfide substituents R were selected to assess the size effect (linear or branched) or potential electronic effect. Pyrrole-2-carbaldehydes 2 were efficiently Pyrrole is a very important structural motif in drug discovery projects because of the wide presence of natural, biologically active pyrrole alkaloid products [25,26]; thus, pyrroleligated 1,3,4-oxadiazole would be a perfect base structure for the development of potential lead compounds [27]. Considering the efficacy of the procedures of constructing a 1,3,4oxadiazole ring, 2-pyrrolyl-5-phenyl-1,3,4-oxadizole 1 would be an ideal core structure, achieved either through dehydration from aroylhydrazide [C] or by cyclization from aroylhydrazone [D]. In the benzene ring, the substituent effects of the core structure 1 on antibacterial activity have been reported for the cases of 4,5-dibromopyrrole [28,29] and 4-nitropyrrole [30], respectively.
Pyrrole-2-carbaldehydes 2, derived from the conversion of D-ribose with L-amino acids [31], were demonstrated to be useful, sustainable platform chemicals for the construction of highly functionalized poly heterocyclic compounds [32]. Since natural amino acids themselves demonstrate specific biological activities [33][34][35], it was envisioned that 1,3,4-oxadiazoles 1 from pyrrole-2-carbaldehydes 2 with the N-amino acid moiety would be very interesting core structures for the investigation of their biological activities. The effect of the amino acid moiety of 1 on antimicrobial activities was screened first, and the substituent effects on the benzene ring were then investigated for 5 and 6 with some selected amino acid moieties. We found a marginal size effect of the alkyl groups from amino acids (Val and Ile, etc.) and superior antibacterial activity of the iodophenol substituents of pyrrole-ligated 1,3,4-oxadiazoles 5 and 6 against S. aureus and A. baumannii. All the syntheses of pyrrole-ligated 1,3,4-oxadiazoles 1, 5, and 6 and their antibacterial activities are closely described herein.

Results and Discussion
Structure and activity relationships (SARs) for pyrrole-ligated 1,3,4-oxadiazoles 1 were generally studied by changing substituent groups in the aromatic rings [28][29][30]. We were interested in the SAR of 1 by N-alkyl substituents because pyrrole-2-carbaldehydes 2, the starting materials for 1,3,4-oxadiazoles 1, are easily prepared from L-amino acids, and each amino acid has its own biological activity. Ten L-amino acids with hydrogen, alkyl, aralkyl, ester, and sulfide substituents R were selected to assess the size effect (linear or branched) or potential electronic effect. Pyrrole-2-carbaldehydes 2 were efficiently prepared by a one-pot ribose conversion with an L-amino methyl ester in the presence of oxalic acid in DMSO, following an improved procedure under 2.5 atm argon at 80 • C [32]. The corresponding pyrrole platform chemicals 2a-2j with the N-amino acid moiety were prepared in yields of 32~63% (Scheme 1 and Table 1). prepared by a one-pot ribose conversion with an L-amino methyl ester in the presence of oxalic acid in DMSO, following an improved procedure under 2.5 atm argon at 80 °C [32]. The corresponding pyrrole platform chemicals 2a-2j with the N-amino acid moiety were prepared in yields of 32~63% (Scheme 1 and Table 1).

Scheme 1.
Preparations of pyrrole-2-carbaldehydes 2, N-benzoylhydrazones 4, and 1,3,4-oxadiazoles 1 from D-ribose conversion with L-amino acids. Two representative procedures are generally utilized for the construction of the 1,3,4oxadizole core, as depicted in Figure 1 [12]. The cyclodehydration route from diacylhydrazine [C] is suitable for pyrrole-2-carboxylic acids [36], whereas the oxidative cyclization route from N-acylhydrazone [D] is widely used for pyrrole-2-carbaldehydes as starting materials [37]. There were various cyclization conditions for 1,3,4-oxadiazoles reported for each conversion [12]. We adopted the oxidative cyclization route of N-acylhydrazones 4, which can be obtained from pyrrole-2-carbaldehydes 2 by condensation with benzohydrazide 3a. The corresponding N-benzoylhydrazones 4a-4j were obtained in decent yields (70~96%) at the reflux temperature of toluene. Oxidative cyclization conditions were then screened using NBS, NIS, and I2 under K2CO3, DBU, Et3N, and NaOH as a base. The condition using NIS/NaOH in DMSO at 100 °C was optimal for providing pyrroleligated 1,3,4-oxadiazoles 1 in yields of 80~98%. It is noteworthy that the NIS-mediated further cyclization of the methylsulfide chain on the pyrrole ring occurred partly for 1j derived from methionine to produce 1k (at a yield of 49%), which explains the lower yield of 1j (50%). All eleven pyrrole-ligated oxadiazoles 1a-1j were rapidly assembled from pyrrole platform chemicals 2 with different amino acid residues and ready for antibacterial assays against Escherichia coli and Staphylococcus aureus as two representative Gram(-) and Gram(+) bacteria (Table 2).  prepared by a one-pot ribose conversion with an L-amino methyl ester in the presence of oxalic acid in DMSO, following an improved procedure under 2.5 atm argon at 80 °C [32]. The corresponding pyrrole platform chemicals 2a-2j with the N-amino acid moiety were prepared in yields of 32~63% (Scheme 1 and Table 1).

Scheme 1.
Preparations of pyrrole-2-carbaldehydes 2, N-benzoylhydrazones 4, and 1,3,4-oxadiazoles 1 from D-ribose conversion with L-amino acids. Two representative procedures are generally utilized for the construction of the 1,3,4oxadizole core, as depicted in Figure 1 [12]. The cyclodehydration route from diacylhydrazine [C] is suitable for pyrrole-2-carboxylic acids [36], whereas the oxidative cyclization route from N-acylhydrazone [D] is widely used for pyrrole-2-carbaldehydes as starting materials [37]. There were various cyclization conditions for 1,3,4-oxadiazoles reported for each conversion [12]. We adopted the oxidative cyclization route of N-acylhydrazones 4, which can be obtained from pyrrole-2-carbaldehydes 2 by condensation with benzohydrazide 3a. The corresponding N-benzoylhydrazones 4a-4j were obtained in decent yields (70~96%) at the reflux temperature of toluene. Oxidative cyclization conditions were then screened using NBS, NIS, and I2 under K2CO3, DBU, Et3N, and NaOH as a base. The condition using NIS/NaOH in DMSO at 100 °C was optimal for providing pyrroleligated 1,3,4-oxadiazoles 1 in yields of 80~98%. It is noteworthy that the NIS-mediated further cyclization of the methylsulfide chain on the pyrrole ring occurred partly for 1j derived from methionine to produce 1k (at a yield of 49%), which explains the lower yield of 1j (50%). All eleven pyrrole-ligated oxadiazoles 1a-1j were rapidly assembled from pyrrole platform chemicals 2 with different amino acid residues and ready for antibacterial assays against Escherichia coli and Staphylococcus aureus as two representative Gram(-) and Gram(+) bacteria (  1 Yields from one-pot reaction [32]. 2 Isolated yields after SiO 2 flash column chromatography. 3 Cyclized product 1k was also obtained in 49% yield. Two representative procedures are generally utilized for the construction of the 1,3,4oxadizole core, as depicted in Figure 1 [12]. The cyclodehydration route from diacylhydrazine [C] is suitable for pyrrole-2-carboxylic acids [36], whereas the oxidative cyclization route from N-acylhydrazone [D] is widely used for pyrrole-2-carbaldehydes as starting materials [37]. There were various cyclization conditions for 1,3,4-oxadiazoles reported for each conversion [12]. We adopted the oxidative cyclization route of N-acylhydrazones 4, which can be obtained from pyrrole-2-carbaldehydes 2 by condensation with benzohydrazide 3a. The corresponding N-benzoylhydrazones 4a-4j were obtained in decent yields (70~96%) at the reflux temperature of toluene. Oxidative cyclization conditions were then screened using NBS, NIS, and I 2 under K 2 CO 3 , DBU, Et 3 N, and NaOH as a base. The condition using NIS/NaOH in DMSO at 100 • C was optimal for providing pyrrole-ligated 1,3,4-oxadiazoles 1 in yields of 80~98%. It is noteworthy that the NIS-mediated further cyclization of the methylsulfide chain on the pyrrole ring occurred partly for 1j derived from methionine to produce 1k (at a yield of 49%), which explains the lower yield of 1j (50%). All eleven pyrrole-ligated oxadiazoles 1a-1j were rapidly assembled from pyrrole platform chemicals 2 with different amino acid residues and ready for antibacterial assays against Escherichia coli and Staphylococcus aureus as two representative Gram(-) and Gram(+) bacteria (Table 2). There was a definite size effect of the alkyl substituent R on antibacterial activity in 1,3,4-oxadiazoles 1. The highest MIC value was required for 1a from glycine (R = H), and it decreased as the size (branch) of the alkyl group increased from alanine (R = Me) to isoleucine (R = s-Bu) (entries 1-5, Table 2). A benzene ring seemed be unimportant, judging from the cases of the benzyl and homobenzyl substituents (entries 6-7). There was no functional group effect for the ester and sulfide, reflecting a lack of electronic interactions between the substituent R and the bacterial enzymes. A comparison of the MIC values for 1j and its cyclized derivative 1k confirmed the importance of the size (or rigidity) effect of R on antibacterial activity (entries 10 and 11). An additional point to mention is that the MIC values for 1 were not much different between Gram(-) and Gram(+) bacteria, indicating that there would be no transport barriers through membranes for these small molecules.
The electronic effects of the substituents on the phenyl ring against antibacterial activities were then investigated for 2-pyrrolyl-5-phenyl-1,3,4-oxadiazoles 5 and 6 with the maximum size effect in the series, derived from valine (R = isopropyl) and isoleucine (R = sec-butyl), respectively. Commercial benzohydrazides 3 with a substituent X of a different electronic nature (e.g., F, Cl, OH, and OMe) were utilized in the synthesis of 5 and 6 (Scheme 2 and Table 3). The condensation reaction of pyrrole-2-carbaldehyde 2c (R = i-Pr) and 2e (R = s-Bu) with various benzohydrazides 3 produced the corresponding N-benzoylhydrazone intermediates in refluxing toluene, which underwent an oxidative cyclization reaction (without purification) to afford 2-pyrrolyl-5-phenyl-1,3,4-oxadiazoles 5 (R = i-Pr) and 6 (R = s-Bu) with various electronic substituents X on the phenyl ring.
SARs of the phenyl substituent X of 2-pyrrolyl-5-phenyl-1,3,4-oxadiazoles 5 and 6 can be deduced from the MIC (µg/mL) in Table 4. ortho-F substitutions provided generally better antibacterial activities than the metaand para-F counterparts (entries 1-3 and 12-14), and chloride was better than fluoride (entries 4 and 15 versus 3 and 14). Iodophenol substituents exhibited superior antibacterial activities against A. baumannii and S. aureus regardless of the position of the hydroxyl substituent (entries 5, 6, 16, and 18). The MIC values of <2 µg/mL for 5f-1 and 8 µg/mL for 6f-1 against A. baumannii were much lower than those of the positive controls (>1024 µg/mL for vancomycin and 128 µg/mL for erythromycin). The "iodophenol effect" on antibacterial activity is obvious when compared with the cases of deiodination products 5f and 6f, the MIC values for which were significantly increased to 128 µg/mL and 512 µg/mL, respectively (entries 7 and 19). The mechanism of the "iodophenol effect" is not clear at present, but it is reasonable to explain that iodide or molecular I 2 may be liberated by the neighboring OH group [39]. No effects or only slight improvements in antibacterial activities were observed for the pyrrole iodination products 5g-1 and 5h-1 from 5g and 5h (entries 8-11), whereas the reverse effect was clear for the pyrrole iodination product 6h-1 from 6h (entries 21 and 22).

Experimental
3.1.1. General Chemical Syntheses 1 H-and 13 C-NMR spectra were recorded on 400 MHz and 100 MHz NMR spectrometers, respectively, in a deuterated solvent (notified in parenthesis) with tetramethylsilane (TMS) as an internal reference. The column chromatography was performed using the method of Still with silica gel 60 and a 70-230 mesh ASTM, using a gradient mixture of EtOAc/hexanes. Reactions were performed in a well-dried flask under an argon atmosphere unless mentioned otherwise.

General Procedure for the Preparation of 1
Formation of Hydrazone 4 from pyrrole-2-carbaldehyde (Step-1): The solution of pyrraline 2 (~1.00 g, 1 equiv.) and benzohydrazide 3a (1 equiv.) in toluene (10 mL) was heated at 110 • C for 6 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with brine and H 2 O, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude product was purified by SiO 2 flash column chromatography to obtain the corresponding benzohydrazone 4.
Step-2: I 2 (1.2 equiv.) and K 2 CO 3 (3 equiv.) were added to a stirred solution of the above imine in 1,4-dioxane (20 mL). The mixture was heated at 85 • C for 6h under an argon atmosphere and cooled to room temperature. The mixture was diluted with CH 2 Cl 2 , washed with a 10% Na 2 S 2 O 3 solution, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. The product was purified by SiO 2 flash column chromatography to obtain pyrrole-fused 1,3,4-oxadiazole 5 or 6.

Biological Evaluation
The minimum inhibitory concentrations (MICs) were determined using the broth microdilution method in a 96-well plate [40,41]. The 96-well plates containing chemicals in two-fold serial dilutions (4 µg/mL to 2048 µg/mL for series 1; 2 µg/mL to 1024 µg/mL for series 5 and 6) were prepared in Luria-Bertani (LB) medium. E. coli, S. aureus, and A. baumannii cells were grown in LB broth to the exponential phase. A 10 µL volume of cells diluted with LB broth to a concentration of 10 8 cells/mL was inoculated on the plates. The MIC was determined after incubation at 37 • C for 16 h under aerobic conditions. The optical density was measured in triple at 600 nm (OD 600 ) using a microplate reader (Bio-Rad, USA) at 20 h after treatment of the chemicals in concentrations of 2,4,8,16,32,64,128,256,512, and 1024 µg/mL. The average and standard deviation values of OD 600 are reported in Table  S1 and Table S2 of the Supporting Information. Vancomycin and Erythromycin were used as positive controls (see Table S2 for the average and standard deviation values of OD 600 ). As the chemicals were dissolved in 100% DMSO, 100% DMSO and triple-distilled water were used as negative controls. The minimum inhibitory concentration (MIC) in Tables 2 and 4 is defined as the lowest concentration of chemicals which provides an average OD 600 value of less than 0.100.

Conclusions
We extended the synthetic utility of pyrrole platform chemicals 2, which can be readily prepared from the sustainable ribose conversion with amino acids, to the pyrrole-ligated 1,3,4-oxadiazole core structure 1 through the reaction with benzohydrazide 3a. The size effect of the R group from the amino acids clearly offered better antibacterial activities for 1,3,4-oxadiazoles 1c and 1e, which were derived from valine and isoleucine, respectively. Benzohydrazides 3 with various electronic X-substituents were utilized for the construction of 2-pyrrolyl-5-phenyl-1,3,4-oxadiazoles 5 and 6 with N-valine and N-isoleucine residues, respectively. Relationships of structure and antibacterial activity were deduced from MIC values for 1,3,4-oxadiazoles 5 and 6 against E. coli, S. aureus and A. baumannii. A positive ortho effect was marginally observed for fluoride substituents. Most importantly, a superior iodophenol effect was evident in the antibacterial activities of 1,3,4-oxadiazoles 5f-1 and 6f-1, which provided much lower MIC values against A. baumannii than those of the vancomycin and erythromycin as positive controls. These findings provide a guiding principle for the design of superior future antimicrobial agents.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/molecules28083638/s1, (1) 1 H/ 13 C-NMR spectra; (2) MIC data against E. coli, S. aureus, and A. baumannii; (3) High-Resolution Mass Spectra for the entire 1,3,4-oxadiazoles synthesized in this paper. Table S1. Determination of MIC for 1 for E. coli and S. aureus by OD600 (20 h). Each value was obtained as an average of at least triple measurements. Table S2. Determination of MIC for 5 and 6 for E. coli, S. aureus, and A. baumannii by OD600 (20 h). Each value was obtained as an average of at least triple measurements (vancomycin and erythromycin as positive controls).